The effect of healthy eating on the development of stomach and colorectal cancer by the smoking and drinking status: Results from the Korean National Cancer Center (KNCC) community cohort study

Abstract Background Determining the effect of dietary factors on cancer is a crucial issue when accounting for the effect of other major risks, such as smoking and drinking. Method A total of 15,563 adults from the Korean National Cancer Center Community Cohort were analyzed to determine and to compare the effect of dietary factors on stomach and colorectal cancer in overall and in the subgroup of non‐smokers (or urinary cotinine concentrations <5 ng/mg) and non‐drinkers with Cox proportional‐hazard models. Results During the mean follow‐up (13.7 years), 469 and 299 cases of stomach and colorectal cancer were identified, respectively. The preventive effect of vegetable, fish, and soybean/tofu intake on colorectal cancer was found in women after adjustment for smoking, drinking, BMI, and sociodemographic factors. In the subgroup analysis of non‐smokers and non‐drinkers, the effect on colorectal cancer was increased in women (≥1 time/week vs. almost never, vegetables: hazard ratio (HR) 0.30, 95% confidence interval (CI) 0.13–0.69; fish: HR 0.46, 95% CI 0.26–0.83), and the fresh fish intake effect on stomach cancer was newly identified in men (HR 0.36, 95% CI 0.15–0.86). These effects were more pronounced and additionally shown in other dietary factors such as soybean or tofu in women and vegetables and fish in men, when subjects with <5 ng/mg urinary cotinine concentrations applied. Conclusion The protective effect of healthy eating on the risk of stomach and colorectal cancer were different by smoking and drinking status. Rigorous control of smoking and drinking effects is necessary when measuring the effect of dietary factors on cancer, properly.

Stomach and colorectal cancer are the most common cancers worldwide; there were more than 1 million new cases of stomach cancer and more than 1.9 million new cases of colorectal cancer in 2020. 1 In Korea, the incidence of stomach and colorectal cancer has been consistently high for the past 20 years: out of 10 cancers in 2020, stomach and colorectal cancer had the second and fourth highest incidence among men and the fifth and third highest incidence among women, respectively. 2any studies have examined the associations among lifestyle factors, such as diet, smoking and drinking, and the prevention, intervention, and risk of stomach and colorectal cancers.The World Cancer Research Fund International has noted the harmful effects of red meat, processed meat, and foods preserved by salting and the beneficial effects of dietary fiber-rich foods. 3Previous studies have also reported that gastric and colorectal cancers show negative associations with soy-based foods, including soybean, tofu, miso, and soymilk, which are prominent in Asian (especially Korean and Japanese) cuisines; 4,5 fruit; and vegetables 6,7 and positive associations with pickled foods, 8,9 fermented soy foods, 4 and red and processed meat. 10,11Moreover, fish intake was found to have no association with stomach cancer risk 12 but a negative association with colorectal cancer risk. 13everal studies have reported differences in the intake of foods and nutrients by smoking and drinking status.Smokers have shown lower intake of fiber, folate, vitamin C, and carotene than never smokers, 14,15 and alcohol intake of more than 10 g/day has been associated with lower intake of fruit, vegetables, dairy products (only among women), and dietary fiber and higher intake of processed meat and salt. 16Even though most previous nutritional epidemiological studies have calculated risk estimates of dietary factors adjusted for smoking and drinking, it is not enough to exclude the interaction or confounding effect of smoking and drinking on dietary factors as those two exposures are substantially correlated with dietary factors even with inconsistent direction.However, to the best of our knowledge, only several cohort studies have evaluated the effect of dietary factors on cancers by smoking status, 17,18 and few have evaluated the effect of dietary factors by drinking status.
Therefore, based on the community-based prospective cohort study, the current study tried to measure the effect of dietary factors on the development of stomach and colorectal cancer, in which the variety of preventive or deleterious effect of dietary factors has been suggested, with the subgroup analysis by the smoking and drinking status as well as statistical adjustment of smoking and drinking in the multiple logistic regression model.As well, urinary cotinine concentration was also applied as an objective measure of smoking exposure together with topography of tobacco use based on the self-report with the structured questionnaire.

| Study population
This study used baseline data obtained from the Korean National Cancer Center (KNCC) Community Cohort study, which recruited adults over 30 years of age living in the community (Sancheong, Uiryeong, Haman, Changwon, Chuncheon, Chungju) between 1993 and 2010.All participants provided written informed consent prior to participating in the study.Baseline data on sociodemographic factors and lifestyle factors, including frequency of food intake, smoking status, and drinking status, were collected by trained interviewers using a questionnaire.Blood and urine samples and anthropometric information were also collected.The design of the KNCC Community Cohort study has been previously published. 19he KNCC Community Cohort study included 16,304 participants at baseline.Of these, 15,563 participants were finally included in the analysis, excluding participants with cancer before enrollment (n = 297) and those under 30 years old (n = 444).

| Assessment of food intake and other variables
The self-administered food frequency questionnaire examined intake of 28 items, including vegetables, fruits, meat, fish, red meat, and soybean/tofu.Participants were asked to share their average intake frequency for each food item using seven categories (almost never, 1 time/ month, 2-3 times/month, 1 time/week, 2-3 times/week, 4-6 times/week, 1 time/day, ≥2 times/day).We grouped these categories into almost never, ≤2-3 times/month, and ≥1 time/week.Based on the results from previous studies, more frequent consumption of vegetables, fruits, soybean or tofu, and fishes, and less frequent consumption of red meats are considered as "healthy eating" in this study.
Age was classified as <50, 50-59, 60-69, or ≥70.Education level was classified as illiterate, middle school, or high school or more.Marital status was classified as single or married.Body mass index (BMI) was determined by dividing body weight by the square of height (kg/m 2 ) and then used to categorize participants as normal weight (<25 kg/m 2 ) or obese (≥25 kg/m 2 ) according to the International Obesity Task Force for adults in the Asia-Pacific region. 20Drinking and smoking status were classified as never, former, or current.Urinary cotinine levels were measured by liquid chromatography-tandem mass spectrometry (using modified methods that have been previously described) 21 and classified as <5 or ≥5 ng/mg. 22The limit of detection for cotinine was 2 ng/ mg.Creatinine levels in the urine were measured via colorimetry (Toshiba 2090 FR; Toshiba, Tokyo, Japan) and used to calculate the creatinine-adjusted cotinine concentration.

| Definition of cancer cases
Cancer cases were identified by linking the cohort data with the Korea Central Cancer Registry data up to the end of 2018 using the personal identification numbers of participants who agreed to the linkage to secondary data, such as the cancer registry.Stomach (C16) and colorectal (C18, C19, C20) cancer were defined using the International Classification of Diseases, 10th revision.

| Statistical analysis
We calculated the mean and standard deviation (SD) for continuous variables.For categorical variables, we calculated prevalence or percentages and used the Chisquare test to analyze the differences among groups.We performed Cox proportional hazard regression analysis to estimate adjusted hazard ratios (HRs), 95% confidence intervals (CIs), and p for trend for risk of stomach and colorectal cancer according to food intake by sex.For each participant, we calculated person-years of follow-up from baseline to diagnosis of cancer, death, or end of follow-up (31 December 2018), whichever occurred first.We used a crude model and two adjusted models: model 1 was adjusted for age, education, marital status, BMI, drinking status, and smoking status, and model 2 was adjusted for age, education, marital status, BMI, drinking status, and urinary cotinine levels.Subgroup analysis was conducted for the never-drinking and never-smoking groups to control for drinking and smoking, which are major risk factors for stomach and colorectal cancer.Because smoking exposure assessed using biomarkers is clearer than that assessed using the questionnaire, we used two subgroups: (1) self-reported never and former smokers and never and former drinkers, and (2) participants with urinary cotinine levels of <5 ng/mg and self-reported never and former drinkers.Additionally, a subgroup analysis was performed for smokers or drinkers: (1) self-reported current smokers or current drinkers, and (2) participants with urinary cotinine levels of ≥5 ng/mg or self-reported current drinkers.
p-values less than 0.05 were considered statistically significant.All analyses were performed using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA).

| RESULTS
A total of 15,563 participants over 30 years of age (men: 6021, women: 9542) were included in the analysis.During a mean follow-up period of 13.7 years, there were 469 stomach cancer cases (men: 296, women: 173) and 299 colorectal cancer cases (men: 148, women: 151).Table 1 presents the baseline characteristics of the study participants stratified by sex.The average age of the participants was approximately 59 years.The education levels were middle school or higher for most men (middle school: 60.8%, high school or more: 27.4%) and middle school or lower for most women (illiterate: 32.3%, middle school: 56.4%).The majority of men had a BMI of <25 kg/m 2 (73.2%) and urinary cotinine of ≥5 ng/mg (57.8%) and were current drinkers (62.2%) and current smokers (48.4%).Most women also had a BMI of <25 kg/m 2 (63.0%) but were never drinkers (80.2%) and never smokers (90.9%) and had a urinary cotinine level of <5 ng/mg (77.6%).Most participants consumed all foods ≥1 time/week, except red meat; approximately 91% of men consumed red meat more than 2-3 times/month, whereas approximately 71% of women consumed it 2-3 times/month or less.
Table 2 presents the HRs (95% CIs) for the associations between food intake frequency and risk of stomach and colorectal cancer after adjustment for age, education level, marital status, BMI, drinking status, and self-reported smoking status (or urinary cotinine level in model 2).Among men, both model 1 and model 2 showed a significant negative dose-response association between soybean/tofu intake and the risk of stomach cancer (model 1: p for trend 0.0014; model 2: p for trend 0.0144).Compared with almost never, a significant association was observed between salted fish intake and increased risk of colorectal cancer in model 1 (≤2-3 times/month: HR 2.04, 95% CI 1.13-3.67;≥1 time/week: HR 1.80, 95% CI 1.01-3.21),but this association was only present for 2-3 times/month in model 2 (HR 1.92, 95% CI 1.05-3.51).Among women, in both models, ≥1 time/week intake of vegetables, fish, and red meat was significantly associated with a reduced risk of colorectal cancer (vegetables: HR 0.32, 95% CI 0.14-0.75;fish: HR 0.46, 95% CI 0.25-0.82;red meat: HR 0.49, 95% CI 0.24-1.00).≥1 time/week intake of soybean/tofu was associated with a reduced risk of colorectal cancer in model 2 (HR 0.45, 95% CI 0.21-0.96),only.No significant associations were observed for stomach cancer.
Table 3 shows the adjusted HRs (95% CI) for the associations between food intake and risk of stomach and colorectal cancer in the subgroup with self-reported non-smokers and non-drinkers.This analysis produced different results compared with the overall analysis (Table 2).Among men, fruit and fresh fish intake had a negative dose-response association for stomach cancer (fruit: p for trend 0.0392; fresh fish: p for trend 0.0324), whereas salted fish intake had a positive dose-response association for colorectal cancer (p for trend 0.0433).Among women, compared with almost never, ≥1 time/ week intake of red meat increased the risk of stomach cancer (HR 2.32, 95% CI 1.07-5.30).More frequent intake of vegetables and fish reduced the risk of colorectal cancer (vegetables: HR 0.30, 95% CI 0.13-0.69;fish: HR 0.46, 95% CI 0.26-0.83).However, the effect of soybean or tofu intake on stomach cancer in men and vegetables intake on stomach cancer in women could not be estimated because no cancer cases were incident in the category of "almost never." Table 4 shows the adjusted HRs (95%CI) for the associations between food intake and risk of cancer in the subgroup of participants with urinary cotinine levels of <5 ng/mg and self-reported never drinkers.Overall, especially in men, the associations were slightly more evident than those in the first subgroup (Table 3), and more foods were significantly associated with cancer risk.Among men, ≥1 time/week intake of vegetables, fish, and fresh fish was strongly associated with stomach cancer risk (vegetables: HR 0.18, 95% CI 0.04-0.81;fish: HR 0.27, 95% CI 0.08-0.87;fresh fish: HR 0.17, 95% CI 0.04-0.69).There was a negative dose-response association between fruit intake and stomach cancer (p for trend 0.0104).Among women, ≤2-3 times/month intake of red meat increased the risk of stomach cancer, whereas ≥1 time/week intake of vegetables, fish, fresh fish, and soybean/tofu was significantly associated with reduced risk of colorectal cancer (vegetables: HR 0.32, 95% CI 0.11-0.88;fish: HR 0.44, 95% CI 0.22-0.87;fresh fish: HR 0.53, 95% CI 0.27-1.04;soybean/tofu: HR 0.41, 95% CI 0.17-0.98).
Tables S1 and S2 show the adjusted HRs (95%CI) for the associations between food intake and risk of cancer in the subgroup of participants with self-reported current smokers (or urinary cotinine levels of ≥5 ng/mg) or current drinkers.Among men, soybean/tofu intake had a negative dose-response association for stomach cancer (subgroup of participants with self-reported current smokers or current drinkers: p for trend 0.0021, subgroup of participants with urinary cotinine levels of ≥5 ng/mg or current drinkers: p for trend 0.0040), which is similar to the analysis results in overall subjects.Among women, negative association between vegetable intake and colorectal cancer incidence was shown (≥1 time/week vs. almost never HR 0.20, 95%CI 0.04-0.99),which is similar with the results from overall subjects and subgroup with non-smokers and non-drinkers.

| DISCUSSION
Our study identified differences in the effects of dietary factors on the risk of stomach and colorectal cancer between the results from the models adjusted for smoking and drinking as confounding factors and the results from subgroups of non-smokers and non-drinkers that restricted these factors.We found that in assessments of smoking exposure, associations evaluated based on urinary cotinine levels were stronger than those evaluated based on self-reported smoking status.In the model adjusted for urinary cotinine levels and other factors, salted fish (men only), soybean/tofu (men and women), vegetable (women only), fish (women only), and red meat (women only) intake were significantly associated with a reduced risk of cancer.However, in the subgroup with self-reported nonsmokers and non-drinkers, fruit (men only), fresh fish (men only), vegetable (women only), and fish (women only) intake were associated with a reduced risk of cancer, and the significant association was more pronounced in the subgroup of never drinkers with urinary cotinine levels of <5 ng/mg.This subgroup also showed a significant association between red meat (women only) and soybean/ tofu (women only) intake and reduced risk of cancer.
Several previous studies have evaluated the effects of dietary factors on stomach and colorectal cancer risk in subgroups restricted to self-reported smoking status, and the effects differed from those adjusted for selfreported smoking status.But few studies have evaluated the effects in subgroups that restricted drinking factors as well.The Shanghai Women's and Men's Health Studies found that intake of all fruits was not associated with distal gastric cancer risk (Q4 vs. Q1 HR 0.79, 95% CI 0.48-1.30,p for trend 0.23), but intake of all fruits except watermelon was associated with a reduced risk of distal gastric cancer in men (Q4 vs. Q1 HR 0.50, 95% CI 0.29-0.84,p for trend 0.004).The significant negative association was maintained in ever smokers (Q4 vs. Q1 HR 0.43, 95% CI 0.26-0.79,p for trend 0.001) but not in never smokers (Q4 vs. Q1 HR 0.77, 95% CI 0.27-2.23,p for trend 0.67). 17In a case-control study, despite considering Helicobacter pylori infection, which was not considered in our study, fruit and vegetable intake was not associated with stomach cancer risk in men (fruit: 108.83 vs. 78.55g/day HR 1.14, 95% CI 0.53-2.46,p for trend 0.736; vegetables: 89.99 vs. 62.97 g/day HR 0.50, 95% CI 0.22-1.12,p for trend 0.094), but never smokers with high fruit and vegetable intake showed a reduced risk of stomach cancer compared with ever smokers with low fruit and vegetable intake (fruit: HR 0.20, 95% CI 0.06-0.65;vegetables: HR 0.25, 95% CI 0.06-0.95). 23imilarly, our study did not find fruit and vegetable intake to be associated with stomach cancer risk in the overall cohort, but it was associated with a reduced risk of stomach cancer among men in all subgroups.
In the European Prospective Investigation into Cancer and Nutrition study, vegetable intake was not associated with colorectal cancer risk (Q5 vs. Q1 HR 0.92, 95% CI 0.79-1.06,p for trend 0.27), except for a negative association among former smokers (Q5 vs. Q1 HR 0.69, 95% CI 0.53-0.89,p for trend <0.01) and a positive association among current smokers (Q5 vs. Q1 HR 1.41, 95% CI 1.03-1.93,p for trend <0.01). 18Our study also found a significant association between vegetable intake and reduced risk of colorectal cancer, including in all subgroups, but only among women.
A cohort study in Korea suggested that soybean/tofu intake was associated with a reduced risk of stomach cancer in women (men: high intake vs. low intake HR 0.77, 95% CI 0.52-1.13;women: high intake vs. low intake HR 0.41, 95% CI 0.22-0.78),but there was no statistically significant association among male smokers and never smokers (p for interaction 0.268). 24Our study showed a significant dose-response association between soybean/ tofu intake and reduced risk of stomach cancer among men, but not in all subgroups.Another cohort study in China and Japan found that dietary isoflavone and soy protein intake were not significantly associated with colorectal cancer risk, but when stratified by smoking status, dietary soy protein intake was significantly associated with a reduced risk of colorectal cancer in ever smokers (Q4 vs. Q1 HR 0.81, 95% CI 0.67-0.99). 5By contrast, our study found a significant association between soybean/ tofu intake and reduced risk of colorectal cancer in the model adjusted for urinary cotinine levels and other factors and among women in the subgroup of never drinkers with urinary cotinine of <5 ng/mg.
Previous studies have evaluated the association between fish intake and the risk of stomach and colorectal cancer, but few have reported the association after restricting smoking and drinking factors.A meta-analysis study suggested that T A B L E 2 Hazard ratios (HRs) and 95% confidence intervals (95% CIs) for stomach and colorectal cancer by frequency of food intake.fish intake was not associated with stomach cancer risk. 12ur study also found no such association; however, among men, fresh fish intake was associated with a reduced risk of stomach cancer in all subgroups.Other systematic review and meta-analysis studies have found a significant association between fish intake and reduced risk of colorectal cancer. 13,25Our study found that this association was significant in women, including in all subgroups.

Total
As such, the beneficial effects of vegetable, fruit, fish, fresh fish, and soybean/tofu intake on stomach and colorectal cancer risk are more evident in subgroups, particularly in men, which can be explained as follows.5][16] In our study, non-smokers and non-drinkers had higher fruit, vegetable (only women), and fresh fish (only women) intake than current smokers and current drinkers (data not shown).Second, a cross-sectional study evaluated the synergistic interaction of smoking and drinking in the reduction of serum carotenoids (lycopene, a-carotene, b-carotene, lutein, b-cryptoxanthin, and zeaxanthin) and found that the average serum carotenoid concentrations did not differ between never smokers and current smokers after adjusting for age  and sex in never drinkers.However, daily alcohol intake reduced the serum carotenoid concentrations (a-carotene, b-carotene, and b-cryptoxanthin) in a dosedependent manner, and these concentrations were obviously lower in current smokers than in never smokers. 26urthermore, another study found that passive smokers had a significantly lower plasma antioxidant status than never smokers, regardless of differences in dietary antioxidant intake. 27revious studies have applied self-reported smoking status to control for smoking factors, but to our knowledge, no study has controlled for smoking exposure using biomarkers.Studies comparing self-reported smoking status and cotinine levels have reported a significantly lower prevalence of self-reported smoking than that assessed by urinary cotinine levels. 28,29oreover, the 2009-2018 Korean National Health and Nutrition Examination Survey showed a discrepancy between self-reported secondhand smoke exposure and that measured by urinary cotinine among never smokers aged 19 or older. 30Therefore, to address the possibility of misclassification by self-reported smoking status, we performed a subgroup analysis to examine both selfreported non-smokers and individuals with urinary cotinine levels less than 5 ng/mg.The preventive effects of fruit, vegetable, fish, and fresh fish intake in the subgroup of never drinkers with urinary cotinine levels less than 5 ng/mg were clearly associated with a reduced risk of cancer compared with the subgroup of self-reported non-smokers and non-drinkers in men, but the significant associations in the two subgroups were similar in women.These results are unsurprising for several reasons.First, the self-reported non-smokers may include hidden smokers, particularly in women.Second, we included former smokers as self-reported never smokers due to small sample sizes, and former smokers who quit abruptly for health or other reasons may have been included.Third, urinary cotinine levels of <5 ng/mg represent a lack of exposure to either secondhand smoke or active smoke, thereby controlling exposure to smoking more completely than the self-reports.In our study, selfreported non-smokers, those with urinary cotinine concentrations of <5 ng/mg, and never drinkers accounted for 51.6%, 42.7%, and 37.8% of men and 82.9%, 77.6%, and 93.4% of women, respectively; thus, differences between the results of the overall cohort and the subgroups may have been more pronounced in men than in women.
Although red meat intake is a well-established risk factor for colorectal cancer, 3 the literature has called for more epidemiologic studies considering Korean dietary culture. 31Our results found a significant association between red meat intake and reduced risk of colorectal cancer in women.Our participants were recruited from the community, mostly from rural areas where meat intake is relatively low.Of the women who participated, 21.7% and 49.0% reported red meat intake of almost never and ≤2-3 times/month, respectively.Thus, we expect that the association is due to the low frequency of red meat intake and the small sample size.
Our study has several limitations.First, food intake data were collected at baseline; changes in dietary habits during the follow-up period were not considered.Second, our data did not include information on food intake in grams per day, menopausal status, and Helicobacter pylori infection, a major risk factor for stomach cancer.Third, we identified 469 stomach cancer cases and 299 colorectal cancer cases during a mean follow-up period of 13.7 years, and the sample sizes of the subgroups of these cases were rather small.As the reason, further analysis on the effect of dietary factors by the smoking and drinking group categorized by amount and duration exposed could not be done, and the effect of some dietary factors were not calculated in women with current smokers or current drinkers (Table S1 and  S2).Fourth, our subjects were 58 years old on average, lived in the community (mostly in rural areas), and had a low frequency of red meat intake (<1 time/week; men: 48.0%, women: 70.7%), so the association of red meat intake with cancer risk could not be effectively evaluated.Despite these limitations, this prospective cohort study identified the different effects of food intake on stomach and colorectal cancer risk by smoking and drinking status, particularly by applying both self-reported smoking status and smoking biomarkers, and confirmed the difference in between two groups.

| CONCLUSION
The current study investigated the effect of vegetable, fruit, fish, and soybean/tofu intake on the risk of stomach and colorectal cancer.The protective effect of healthy eating on the risk of those cancer were identified but different by smoking and drinking status.The effect was more pronounced in the subgroup of non-drinkers with <5 ng/mg urinary cotinine concentrations than among non-drinkers and self-reported non-smokers.
Rigorous control of smoking and drinking effects looks necessary when measuring the effect of dietary factors on cancer, properly, although further study is in need to confirm the current study results with the lager sample followed up longer period.

T A B L E 1
Baseline characteristics of study participants.
a Crude.b Adjusted for age, education, marital status, smoking status, drinking status, BMI.c

95% CI) a p for trend HR (95% CI) b
Hazard ratios (HRs) and 95% confidence intervals (95% CIs) for stomach and colorectal cancer by frequency of food intake among self-reported non-smokers and non-drinkers.
T A B L E 3

95% CI) a p for trend HR (95% CI) b
Hazard ratios (HRs) and 95% confidence intervals (95% CIs) for stomach and colorectal cancer by frequency of food intake among urinary cotinine level a Crude.b Adjusted for age, education, marital status, BMI.T A B L E 3 (Continued) T A B L E 4 a Crude.b Adjusted for age, education, marital status, BMI.